EP1186295B1 - Pharmaceutical preparations containing extracts of soy isoflavone and probiotic micro-organisms - Google Patents

Abstract

Pharmaceutical preparations containing soja extracts rich in isoflavones and living probiotic micro-organisms, given simultaneously, separately or sequentially for treatment of menopausal symptoms.

Description

The present invention relates to associations between soy isoflavones and probiotic microorganisms, and their use in therapy.

Soy belongs to the Fabaceae family . The fruit of soy is a pod containing rounded or ovoid seeds enclosed by two, three or four. These soybeans are composed mainly of carbohydrates, proteins and fats. Carbohydrates, which represent 15 to 25% of the material consist of simple sugars such as sucrose, raffinose and stachyose, pentosames and galactosannes, but very little starch. Proteins are abundant since they represent 35 to 40% of the material and consist mainly of albumin and globulin. It is this high protein content that makes the nutritional value of soybeans. The lipids correspond to 15 to 20% of the material and consist mainly of glycerides of saturated or unsaturated fatty acids, an unsaponifiable rich in phytosterols and a polar lipid fraction rich in phospholipids called lecithin.

Isoflavones are also an important constituent of soybean seeds, but the soy content of isoflavones varies considerably depending on the species, the maturity of the plant and the conditions of cultivation. These isoflavones are heterocyclic polyphenols characterized by a sequence of C15 Ar-C ₃ -Ar type arranged in a 1,2-diphenylpropane pattern. The general structure is given in formula (1) below:

et daidzéine de formule (3) où R₇ = hydrogène,

sont présentes sous différentes formes: glycosides, malonides, acétyles et aglycones, cette dernière forme étant minoritaire puisqu'elle ne représente que 1 à 2% des isoflavones totaux. Les conjugaisons ont lieu en position 7.In the plant, the main isoflavones, genistein of formula (2)

and daidzein of formula (3) wherein R ₇ = hydrogen,

are present in different forms: glycosides, malonides, acetyls and aglycones, the latter being a minority form since it represents only 1 to 2% of the total isoflavones. The conjugations take place in position 7.

Wang H. et al . ( J. Agric ., Food Chem. (1994) 42, No. 8, pages 1666 to 1673) determined the presence of 2,275 mg of isoflavones, per gram of soybean, in all the forms mentioned above, which corresponds to contents 43% in daidzein and 57% in genistein.

Tham DM et al. ( J. Clin Endocrinol Metab. (1998) 83 , No. 7, pages 2232 to 2235) titrated two different species at 1.005 mg / g and 4.031 mg / g with 36% and 32%, respectively, of daidzein.

Patent abstract of Japan, vol. 1998 No. 1, January 30, 1998, JP 09238647 mentions a food capable of preventing cancer and containing isoflavones obtained by hydrolysis of isoflavones glycosides contained in soy milk by the action of microorganisms on said soy milk, said microorganisms belonging to the genus Lactobacillus, Bifidobacterium, Streptococcus, Bacillus, Sacharomyces, Torulaspora and Candida.

International application WO 00/03684 describes soy formulations whose isoflavone concentration is higher than that contained in the natural product. These formulations may optionally comprise a medicament, such as, for example, hormone replacement treatments, as well as various other elements such as, for example, digestion enzymes or vitamins, but there is never any mention of microorganisms.

These compositions reduce the effects of menopause, including hot flushes (page 18 lines 16 to 17 + page 19 line 11 to page 20 line 12).

The international application WO 99/07239 describes a combination of fructo-oligosaccharide, soya flour containing flavonoids and ground flax seeds containing lignans, able to increase the intestinal activity and to facilitate the conversion of phytoestrogens into forms that can be used by the intestines, as well as their absorption. In this combination, fructo-oligosaccharides are used for their prebiotic effects, i.e. by their ability to selectively increase the growth of bifidobacteria and lactobacilli that increase acid production in the intestines. These compositions are useful for treating the symptoms of menopause.

The international application WO 98/21945 describes compositions which are capable of reducing the manifestations of SPRT (Symptoms of Persistent Reproductive Transition), among which flushing, of heat; these compositions contain phyto-estrogen, in particular from soy associated with sugars and cholines.

In fact, in general, the value of 2 mg of isoflavones per gram of soybeans in the dry seed is retained.

The structure of isoflavones has homologies with those of estrogens. Thus, without being steroids, genistein and daidzein possess hydroxyl groups at the 7 'and 4' position in a stereochemical configuration analogous to that of the hydroxyls of the estradiol molecule of formula (4).

Because of this structural homology with estradiol, and more particularly because of the distance and the dihedral angle between the two hydroxyl groups, the isoflavones have an estrogenic activity. They can indeed bind as estradiol on type II (β) receptors (Bennetau-Pelissero Scientific Letter of the French Institute for Nutrition (1997) 47 ), however, this activity is at least 500-fold lower than that of estradiol.

Indeed, if the activity of estradiol is set at 100, then daidzein has an activity of 0.013, genistein has an activity of 0.084 and equol, the metabolic derivative of daidzein, has an activity of 0, 61 (Markiewicz L., J. Steroid Biochem Molec Biol (1993) 45 , 399-405).

• 4 à 10 fois moins de probabilités de décéder d'un cancer hormono-dépendant (sein, endomètre, prostate et colon),
• moins de symptômes corrélés à des désordres hormonaux au moment de la ménopause (ostéoporose, bouffées de chaleur), et
• moins de maladies coronariennes et du coeur.

Soy is consumed by Asian populations, especially in fermented forms such as miso or tempeh. The observation of these populations, which therefore have a diet very rich in phytoestrogens and consume between 20 and 150 mg of isoflavones per day Murkies A. et al . ( J. Clin Endocrinol Metab . (1998) 83 , No. 2, pp . 297-303 ) revealed differences in health status in Western populations. These populations present:

• 4 to 10 times less likely to die of hormone-dependent cancer (breast, endometrium, prostate and colon),
• fewer symptoms correlated with hormonal disorders at the time of menopause (osteoporosis, hot flashes), and
• less coronary heart disease and heart.

Because of their structural homology with estrogens, isoflavones are often used in medicine, mainly in medical gynecology. Thus, Wilcox C. et al. ( BMJ (1990) 24 , pages 1757-1761) and Washburn SA et al . (Third International Conference on Phyto-oestrogens (1995) Little Rock, USA) showed respectively in controlled studies against placebo, a significant and significant decrease in hot flashes when ingesting soybeans at a rate of 40 g / day for 6 weeks. Albertazzi P. et al . (Am J. Obstet, Gynecol, (1998) 91 , pp. 6-11) confirmed this effect with 60 g of soybean (76 mg of isoflavones) per day in a randomized study in more than 100 postmenopausal women. These studies therefore demonstrate the effect of phytoestrogens on hot flushes.

In addition, in women, studies have been conducted by Valente M. et al. ( Tissue Calc Int (1994) 54 , pages 377-380) with ipriflavone, a synthetic prodrug of daidzein, at 500-600 mg per day and showed in 40 postmenopausal women with osteoporosis a bone at L1-L5 with a return to normal in 12 months. Gambacciani M. et al. ( Bone and mineral, 26, pages 19-26) has shown that bone loss can also be avoided in women on Luteinizing Hormone Releasing Hormone (LHRH) agonists . Blair et al. ( J. Cell Biochem., 1996, 61, pages 629 to 637) explain this action by demonstrating an inhibition of osteoclast activity by genistein.

In the case of hormone-dependent cancers, Barnes S. et al . ( Diet and cancer: markers , prevention and treatment (1994) Plenum Press, pages 135-147) showed a reduction in the number and size of tumors. Basic studies by Pagliacci NC et al . (European J. of Cancer (1994) 30A (11) pages 1675 to 1682) have shown inhibition of human breast cancer cells by phytoestrogens, while Wutke W. (in Proceedings of 2 ^nd Congress European Society of Gynecology (1997)) has demonstrated the inhibition of the expression of genes responsible for the growth of cell cultures. Finally, the study by Lee HP et al. ( Lancet (1991) 337 , No. 18, pages 1197 to 1200) has shown that the risk of breast cancer falls with a high-soy diet in postmenopausal women.

The small intestine is the preferred site of isoflavone absorption because the contact area is large, and the blood network dense. However, only the aglycone forms (genistein and daidzein) are absorbed because the glycosides have too much hydrophilicity, and too much molecular weight.

It has been demonstrated that only aglycone forms have estrogenic activity in animals (Tham DM et al., Cited above, and Moreau L. et al ., References in Obstetric Gynecology (1999), pages 229-234, Springer-Verlag). The biotransformation of glycosylated isoflavones into equol, genistein and daidzein is therefore necessary for the observation of the beneficial effects of soy.

Chang YC et al. ( J. Nat Prod (1995) 58, No. 12, pages 1892-1896) have shown that this necessary transformation of glycosylated isoflavones (daidzin and genistin) into daidzein, genistein and equol is carried out by the intestinal microflora.

In order to use the isoflavones, the intestinal flora therefore comprises bacteria capable of hydrolyzing the β 1-4 linkages and thus releasing the aglycone forms. These microorganisms secrete enzymes such as β-glucosidases, but this deglycosylation is also carried out partly by simple hydrolysis with gastric hydrochloric acid.

Finally, the molecules already absorbed, and therefore conjugated, can be deconjugated and reabsorbed thanks to the enzymes β-glucuronidase and arylsulfurase.

The diversity of the intestinal microflora plays an important role in the metabolic pathways and bioavailability of isoflavones, including Lactobacilli, Bacteroides and Bifidobacteria (Xu X. et al ., (1995) Bioavailability of soybean isoflavons depends on gut microflora in women I, Nutro 125, 2307-2315).

In general, the conversion is effective, and only 14 to 43% of ingested isoflavones are found intact in the urine.

However, there is a large individual metabolic variety and Moreau et al . (References in obstetric gynecology already cited) have shown that 30% of the population does not have the flora capable of metabolizing the most effective equol - aglycone form compared with estrogenic effects, making it difficult to treat with isoflavones.

• Effets sur le tube digestif: amélioration de la digestion du lactose chez les personnes déficientes en lactase, prévention des troubles intestinaux, adhérence aux cultures de cellules intestinales, stimulation de l'immunité intestinales dans les modèles animaux, stabilisation de la maladie de Crohn et régulation de la motilité intestinale.
• Modification de la microflore: équilibre des bactéries intestinales, augmentation des bactéries fécales, diminution de l'activité des enzymes fécales, diminution de la mutagénicité fécale, soulagement des symptômes de l'intolérance au lactose et colonisation du tube digestif.
• Effet sur la diarrhée: prévention et/ou traitement des diarrhées aiguës et à rotavirus, réduction des diarrhées à rotavirus, prévention de la diarrhée associée aux antibiotiques, traitement des diarrhées récidivantes à Clostridium difficile et traitement des diarrhées persistantes chez l'enfant.
• Effets systémiques: stimulation de l'activité phagocytaire, stimulation de la production d'interféron γ par les cultures de cellules mononucléaires du sang humain, réduction de l'hypertension dans les modèles animaux et humains, effets bénéfiques sur la cancer superficiel de la vessie et soulagement des symptômes cliniques chez les enfants atteints de dermatite optique.
• Effets physiologiques: production de bactériocines et effet antagoniste contre Helicobacter pylori.

Probiotics are "living organisms that, when ingested in sufficient quantity, exert beneficial health effects that go beyond the inherent nutritional virtues". The health benefits are not limited to improving the microbial balance in the gut, but may include other effects. Thus, for lactic acid bacteria, five types of effects are observed:

• Effects on the digestive tract: improvement of lactose digestion in people with lactase deficiency, prevention of intestinal disorders, adhesion to intestinal cell cultures, stimulation of intestinal immunity in animal models, stabilization of Crohn's disease and regulation of intestinal motility.
• Modification of microflora: balance of intestinal bacteria, increase of fecal bacteria, decrease of fecal enzyme activity, decrease of faecal mutagenicity, relief of symptoms of lactose intolerance and colonization of the digestive tract.
• Effect on diarrhea: prevention and / or treatment of acute and rotavirus diarrhea, reduction of rotavirus diarrhea, prevention of antibiotic-associated diarrhea, treatment of recurrent Clostridium difficile diarrhea and treatment of persistent diarrhea in children.
• Systemic effects: stimulation of phagocytic activity, stimulation of γ-interferon production by mononuclear cultures of human blood, reduction of hypertension in animal and human models, beneficial effects on superficial bladder cancer and relief of clinical symptoms in children with optic dermatitis.
• Physiological effects: production of bacteriocins and antagonistic effect against Helicobacter pylori.

Bifidobacteria, in turn, improve gastrointestinal transit, decrease the risk of colon cancer and have antimicrobial activity and immunomodulatory effects, while Lactobacillus acidophilus has hypocholesterolemic, antimicrobial and immunomodulatory effects, while preventing breast cancer. colon.

Consequently, it appears necessary to overcome the low estrogenic activity of isoflavones and improve their activity in patients with pre- and / or menopausal symptoms and more particularly in patients with little or no response.

However, the inventors have surprisingly found that certain probiotic microorganisms associated with isoflavones could solve this problem.

The subject of the present invention is therefore pharmaceutical preparations comprising extracts titrated with soy isoflavones and live probiotic microorganisms, as a combination product for simultaneous, separate or spread administration in time for the treatment of symptoms related to menopause.

In a particular embodiment, the isoflavone extract is titrated with 10 to 50% isoflavones.

In another particular embodiment, the probiotic microorganisms are chosen from the group consisting of Enterococcus faecium, Propionibacterium acidipropionici, Lactobacillus acidophilus , Lactobacillus brevis, Lactobacillus plantarum , Lactobacillus casei, Lactobacillus rhamnosus , Pediococcus acidilactici and Streptococcus thermophilus and mixtures of these microorganisms.

In a particularly advantageous embodiment of the invention, the preparations comprise the isoflavone extract titrated between 10 and 50% isoflavones and the probiotic microorganisms are selected from the group consisting of Enterococcus faecium , Propionibacterium acidipropionici , Lactobacillus acidophilus and Lactobacillus brevis.

For the purposes of the present invention, the term "titrated extract" is intended to mean soybean isoflavones, an extract prepared as follows: the crushed soybean seeds are extracted with an aqueous-alcoholic solution. After removal of the alcohol, the aqueous solution is delipidated with an organic solvent and then column-purified on an absorbent resin.

Probiotic microorganisms are chosen on the basis of their biological properties and their ability to metabolize isoflavones.

These microorganisms may be used in commercially available culture form, in the form of a microbial suspension or preferably in the form of a lyophilizate prepared under standard conditions known to those skilled in the art.

In a particularly advantageous embodiment of the invention, these microorganisms are present at a rate of 50 × 10 ⁹ to 1.10 ⁶ bacteria per unit dose and the isoflavone extract is present in a proportion of 100 to 300 mg per unit of intake. .

The preparations according to the invention are particularly useful as a medicament, in particular for the treatment of symptoms related to menopause, particularly against hot flushes.

The preparations according to the invention comprising the soy extract and the microorganisms may be presented in any dry or fluid form usually used, in particular in the form of tablets, tablets, capsules, capsules or oral solutions in combination. with any pharmaceutically acceptable excipient.

The isoflavone extract and probiotic microorganisms may also be administered as separate unit doses, including tablets, tablets, capsules, capsules or oral solutions that may be administered separately or simultaneously or in a staggered manner. in time.

The preparations according to the invention allow a metabolization of glycosylated forms into active aglycones from the first absorption, at the level of the duodenum, the maximum absorption zone of the digestive tract.

The following examples illustrate the invention without limiting it.

Example 1: Formulation Examples Formulation 1:

• extrait de soja titré à 15% en isoflavones, 250 mg dans une gélule de taille 1,
• lyophilisat de Lactobacillus acidophilus, 10⁹ bactéries dans une gélule de taille 1

prendre 1 gélule de chaque simultanément par jour.

• soybean extract 15% isoflavones, 250 mg in a size 1 capsule,
• lyophilizate of Lactobacillus acidophilus , 10 ⁹ bacteria in a size 1 capsule

take 1 capsule of each simultaneously per day.

Formulation 2:

• extrait de soja titré à 15% en isoflavones, 100 mg,
• lyophilisat de Lactobacillus acidophilus, 10⁶ bactéries dans même une gélule de taille 1

prendre 2 gélules par jour matin et soir ou 2 le matin ou 2 le soir.

• soybean extract titrated to 15% isoflavones, 100 mg,
• lyophilizate of Lactobacillus acidophilus , 10 ⁶ bacteria in same size 1 capsule

take 2 capsules a day morning and evening or 2 in the morning or 2 in the evening.

Formulation 3:

• extrait de soja titré à 40% en isoflavones, 200 mg formulé dans un comprimé,
• lyophilisat de Lactobacillus brevis, 10⁹ bactéries dans une gélule de taille 1

prendre une gélule de chaque simultanément par jour.

• soybean extract, 40% isoflavones, 200 mg formulated in a tablet,
• Lyophilisate of Lactobacillus brevis, 10 ⁹ bacteria in a size 1 capsule

take one capsule of each simultaneously a day.

Formulation 4:

• extrait de soja titré à 10% en isoflavones, 200 mg dans une capsule,
• lyophilisat de Enterococus faecium, 10⁹ bactéries dans une capsule

prendre 4 capsules de chaque simultanément par jour.

• soybean extract titrated at 10% isoflavones, 200 mg in a capsule,
• lyophilisate of Enterococus faecium, 10 ⁹ bacteria in one capsule

take 4 capsules of each simultaneously a day.

Formulation 5:

• solution hydro-alcoolique à 2% en extrait de soja titré à 50% en isoflavones,
• suspension microbienne de Lactobacillus acidophilus à 10⁸ germes/g,

prendre à l'aide d'une cuillère à mesure 10 ml de chacune des solutions par jour.

• 2% hydroalcoholic solution in soybean extract with 50% isoflavone content,
• microbial suspension of Lactobacillus acidophilus at 10 ⁸ germs / g,

take 10 ml each of the solutions per day with a spoon.

Example 2 Selection of Isoflavone Metabolizing Microorganisms 1) Operating mode:

Microorganisms, present in the faecal flora and capable of metabolizing genistin and daidzine respectively in genistein and daidzein, were cultured for 24, 48 and 72 hours in a nutrient medium containing: meat peptone 10 g / l , meat extract 10 g / l, yeast extract 5 g / l, glucose 20 g / l, sodium acetate 5 g / l, tween80 1 g / l, dipotassium phosphate 2 g / l, ammonium citrate 2 g magnesium sulphate 0.2 g / l, manganese sulphate 0.05 g / l, and a soy extract. At each of these times, a microbial culture sample is taken, diluted by half in methanol and then filtered. The methanolic solution is analyzed in high performance liquid chromatography. Daidzin, genistin, daidzein and genistein are assayed by high performance liquid chromatography in the starting extract and in the media metabolized by the different microorganisms. The contents of the aglycone and glycosylated forms of the metabolized extract are thus calculated with respect to the glycosylated forms (100%) in the initial extract.

2) Results.

They are summarized in Table 1 below. TABLE 1 Enterococcus faecium Propionibacterium acidipropionici Lactobacillus acidophilus Lactobacillus brevis Lactobacillus plantarum Pediococcus acidilactici Streptococcus thermophilus Lactobacillus casei Lactobacillus rhamnosus genistin 5.5% 12% 6.5% 2% 53% 63% 73% 85% 5% daidzin 0.1% 33% 16% 16% 75% 77% 65% 80% 26% genistein 80% 72% 83% 81% 44% 30% 25% 13% 85% daidzein 85% 57% 84% 82% 22% 23% 33% 18% 71%

These microorganism strains metabolize approximately 80% of the glycoconjugated forms of isoflavones into aglycone forms that are readily absorbed in the intestine; Enterococcus faecium , Propionibacterium acidipropionici , Lactobacillus acidophilus and Lactobacillus brevi are the most active strains.

Example 3: Clinical Evaluation 1) Modalities of the study:

It was implemented in order to test the efficacy of the soy-probiotic combination with the criterion of hot flash efficacy in the menopausal indication with 2 groups of menopausal women receiving a soy extract associated or not to the probiotic.

This study was conducted for 3 months with a pre-inclusion period of one month.

A group of 50 menopausal women were treated once a day with a capsule containing 10 ⁹ bacteria / g of Lactobacillus acidophilus . Another group of 50 menopausal women received once daily a 250 mg capsule of 15% soybean extract with isoflavones and one placebo capsule.

Each subject recorded in a self-report the number of daily hot flashes and their intensity according to a pre-established scale.

2) Results:

This study shows the important positive role of probiotics in the regularity and intensity of the response to phytoestrogens, since the preparations according to the invention allow women who have received the soy extract associated with the probiotic to note a decrease in the number and intensity of hot flushes greater than those who received the soy extract alone.

Claims (6)

1. Use of a pharmaceutical preparation containing extracts titrated with soy isoflavones and live probiotic micro-organisms, as a combination product for simultaneous, separate or time-released administration for the preparation of a medicinal product, useful in the treatment of hot flashes related to menopause, characterised in that the probiotic micro-organisms are selected from the group comprising Enterococcus faecium, Propionibacterium acidipropionici, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Pediococcus acidilactici and Streptococcus thermophilus and mixtures of these micro-organisms.
2. Use as claimed in claim 1, characterised in that the isoflavone extract is titrated between 10% and 50% isoflavones.
3. Use as claimed in claim 1 and 2, characterised in that the isoflavone extract is titrated between 10% and 50% isoflavones and the probiotic micro-organisms are selected from the group comprising Enterococcus faecium, Propionibacterium acidipropionici, Lactobacillus acidophilus and Lactobacillus brevis.
4. Use as claimed in any of claims 1 to 3, characterised in that the probiotic micro-organisms are used in the form of a microbial suspension or lyophilizate.
5. Use as claimed in any of claims 1 to 4, characterised in that the probiotic micro-organisms are present in a concentration between 50.10⁹ and 1.10⁶ bacteria per dosage unit.
6. Use as claimed in any of claims 1 to 5, characterised in that the isoflavone extract is present at a rate of 100 to 300 mg per dosage unit.